Purpose: Non-invasive techniques to monitor the survival and migration of transplanted stem cells in real-time is crucial for the success of stem cell therapy. The aim of this study was to explore multimodality molecular imaging to monitor transplanted stem cells with a triple-fused reporter gene [TGF; herpes simplex virus type 1 thymidine kinase (HSV1-tk), enhanced green fluorescence protein (eGFP), and firefly luciferase (FLuc)] in acute myocardial infarction rat models. Methods: Rat myocardial infarction was established by ligating the left anterior descending coronary artery. A recombinant adenovirus carrying TGF (Ad5-TGF) was constructed. After transfection with Ad5-TGF, 5×106 bone marrow mesenchymal stem cells (BMSCs) were transplanted into the anterior wall of the left ventricle (n = 14). Untransfected BMSCs were as controls (n = 8). MicroPET/CT, fluorescence and bioluminescence imaging were performed. Continuous images were obtained at day 2, 3 and 7 after transplantation with all three imaging modalities and additional images were performed with bioluminescence imaging until day 15 after transplantation. Results: High signals in the heart area were observed using microPET/CT, fluorescence and bioluminescence imaging of infarcted rats injected with Ad5-TGF-transfected BMSCs, whereas no signals were observed in controls. Semi-quantitative analysis showed the gradual decrease of signals in all three imaging modalities with time. Immunohistochemistry assays confirmed the location of the TGF protein expression was the same as the site of stem cell-specific marker expression, suggesting that TGF tracked the stem cells in situ. Conclusions: We demonstrated that TGF could be used as a reporter gene to monitor stem cells in a myocardial infarction model by multimodality molecular imaging.